The role of metabolism in the pathogenesis of vitamin D3 deficiency and variable responses to vitamin D3 treatment in human colorectal cancer is yet uncertain. Vitamin D3 is metabolized via CYP-P450 catalyzed hydroxylation reactions. The three potential quantitative measures of vitamin D3 hydroxylating enzymes are: 25-D3/D3 ratio ` a measure of CYP27A1 and CYP2R1 catalyzed hydroxylation of D3 to 25-D3;1,25-D3/25-D3 ratio `a measure of CYP27B1 catalyzed hydroxylation of 25-D3 to 1,25-D3;and 24,25-D3/25-D3 ratio ` a measure of the predominantly CYP24A1 catalyzed hydroxylation of 25-D3 to 24,25-D3. Our preliminary studies showed significant changes between day 90 and baseline 25-D3/D3 ratio (p<0.0001) and 1,25-D3 /25-D3 ratio (p= 0.0241) in 30 vitamin D3 deficient colorectal cancer (CRC) patients. Furthermore, genotyping studies of the 74 tagSNPs in the key vitamin D3 metabolism genes (DBP, VDR, CYP27A1, CYP27B1 and CYP24A1) revealed a significant association between rs4588 polymorphism, a non-synonymous coding SNP in the DBP gene and serum 25-D3 levels (1 =0.0001). Therefore, we hypothesize that functionally relevant polymorphisms (SNPs) in genes involved in Vitamin D3 biotransformation may play a role in an individual's vitamin D3 status and that these SNPs may explain variable response to Vitamin D3 supplementation. This hypothesis will be tested in the two specific aims below using blood samples from two CRC patient cohorts (non intervention and cholecalciferol intervention of colorectal cancer patients). Aim 1a): determine the baseline vitamin D3 status (activating and inactivating enzyme hydroxylation) in 282 non- intervention and 50 intervention patients 1b): determine the effect of intervention with 2000 IU daily oral cholecalciferol supplementation on vitamin D3 status and hydroxylation phenotypes at the 3 month time point for the 50 patients. The endpoint measures for this specific aim are: (i) Total serum 25-D3;24,25-D3 and 1,25- D3 levels (ii) serum 25-D3/D3;1,25-D3/25-D3 and 24,25-D3/25-D3 ratios, compared to the standard measure of serum 25-D3 only. (iii) Free serum index of 25-D3, 24,25-D3 and 1,25-D3 levels (measured as free index of each vitamin D3 metabolite---defined as the molar ratio of the total serum metabolite/DBP). Aim 2: Genotype for 74 tagSNPs identified in the five key genes (DBP, VDR, CYP27A1, CYP27B1 and CYP24A1) that regulate vitamin D3 metabolism, using genomic DNA isolated from blood samples of all 282 non- intervention and 50 cholecalciferol supplemented CRC patients. The genotyping data will be used to evaluate the correlation between the tagSNPs, baseline vitamin D3 status/hydroxylation phenotypes and demographic covariates for (a) non-intervention cohort of 282 CRC patients and (b) an intervention cohort of 50 CRC patients (before and after cholecalciferol supplementation). If successful, this proposal may help to explain in part the variations that lead to vitamin D3 deficiency. The study may also serve as a paradigm to predict the efficacy of vitamin D3-based interventions. PUBLIC HEALTH RELEVANCE: Vitamin D deficiency and its relationship to cancer and other chronic diseases is an important and growing public health problem. In this proposal we seek to find blood vitamin Dmetabolite patterns and inherited changes in the vitamin D metabolism genes related to vitamin D deficiency. The information derived from this study could be used to identify individuals at high risk of developing diseases associated with vitamin D3 deficiency and to also predict responses to vitamin D treatment.